Glucose detection in blood and urine is necessary for the diagnosis of diabetes. Glucose monitoring in fermentation of food industry is also necessary since the amount of glucose in the fermentation greatly influences the quality of the food products. See G. Harsanyi, “Sensors in Biomedical Applications: Fundamentals, Technology and Applications,” Technomic Pub., Lancaster, Pa. (2000)). Glucose oxidase (GOD) has been widely used in glucose biosensors due to its high selectivity for glucose and high activity over a broad range of pH values. See B. J. White et al., Biochem. Biophys. Res. Commun., 296, 1069 (2002). Sensitivity and stability of a glucose biosensor are key features for its quantitative analysis applications. See, e.g., V. G. Gavalas et al., Analyt. Chim. Acta, 67, 404 (2000); M. Delvaux et al., Biosens. Bioelectron., 18, 943 (2003). Many attempts have been made to improve the features of the biosensors, including making use of novel immobilization techniques and new enzyme immobilization materials. Glassy carbon (GC), graphite, carbon paste, carbon fibers, porous carbon, and carbon spheres are commonly used as electrode materials for biosensor immobilization matrices (M. Albareda-Sirvent et al., Sens. Actuat., B69, 153 (2000); S. Sotriropoulou et al., Biosens. Bioelectron., 18, 211 (2003). Some GOD sensors do exhibit high sensitivity. However, the lifetime of the biosensors is only a few weeks and the stability is low, thus limiting their utility in harsh environments. See Z. Liron et al., eds., Novel Approaches in Biosensors and Rapid Diagnostic Assays, Kluwer, Acad./Plenum Pub., NY (2001) at page 203.
Carbon nanotubes (CNTs) are a new type of carbon material that can be considered to result from folding graphene layers into carbon cylinders. CNTs can be composed of a single shell-single-walled nanotubes (SWNTs) or several shells-multi-walled nanotubes (MWNTs). See S. Iijima et al., Nature, 363, 603 (1993); S. Iijima, Nature, 354, 56 (1991). CNTs have attracted increasing interest for potential applications in electron field emitters, field-effect transistors, actuators, and gas sensors because of their special geometry and unique electronic, mechanical, chemical, and thermal properties. CNTs have been recognized as promising electrode materials. SWNTs are semiconductors that exhibit high mobility since all their atoms are located on the tube surface. Only recently, have CNTs been investigated as biosensors for glucose and DNA detection and their performance has been found to be much superior to those of other carbon electrodes in terms of reaction rate, reversibility, and detection limit. See, e.g., S. Sotriropoulou et al., Anal. Bioanal. Chem., 375, 103 (2003); A. Guiseppe-Elie et al., Nanotech., 13, 559 (2002); M. L. Pedano et al., Biosens. Bioelectron., 18, 269 (2003); K. Bestman et al., Nano Lett., 3, 727 (2003); M. Gao et al., Synth. Metals 137, 1393 (2003). However, their potential utility has been limited by the need to functionalize the surface of the tubes to a sufficient stability and density, either covalently or noncovalently, while not disrupting the nanotube π delocalized system.
Chen et al., J. Amer. Chem. Soc., 123, 3838 (2001) immobilized the protein ferritin on SWNTs via a 1-pyrenebutanoic acid, succinimidyl ester linking group. The linking group was noncovalently adsorbed onto the walls of SWNTs by π-stacking. The amine groups on the protein reacted with the anchored succinimidyl ester to form amide bonds that can immobilize proteins or other molecules containing free NH2 groups. However, Chen et al. did not report the electrical characteristics of the functionalized SWNTs.
K. Besterman et al., Nano Lett., 3, 727 (2003) used the same linking group to bind the enzyme glucose oxidase, E.C1.1.3.4, on carbon nanotubes. They observed that immobilization of the enzyme decreased the conductance of the SWNTs. Using a standard reference electrode, the conductance of the functionalized SWNTs were found to be sensitive to changes in pH and to glucose concentration. However, to yield useful nanoscale biosensors, it will be necessary to increase the effective density of sensing molecules on the surface of the SWNTs.
V. M. Mirsky et al., Biosensors & Bioelectronics, 12, 977 (1997) reported that self-assembled monolayers of functionalized thiols could be assembled on gold electrodes and employed to immobilize antibodies to human serum albumin (HSA). Subsequent binding of HSA led to a decrease of the electrode capacitance. While promising for use in the fabrication of conventional electrodes, this approach has not been applied to fabricate nanosensors. Thus, a continuing need exists for methods to prepare durable, sensitive nanosensors for biological analytes.